Ischemia With Preconditioning in Wistar Rats Maintains Mitochondrial Respiration, Even With Mild Hepatocellular Disturbance.

INTRODUCTION: In hepatectomy or liver transplantation, preconditioning is a procedure indicated to protect the organ from ischemia-reperfusion injury (I-R). OBJECTIVE: Evaluate the effect of preconditioning after hepatic I-R in Wistar rats, through mitochondrial respiration, liver histology, and profile. METHOD: Twenty male Wistar rats, weighing on average 307.1 g, were anesthetized with sodium thiopental (25 mg/kg) intravenously and xylazine hydrochloride (30 mg/kg) intramuscularly. The animals were divided into 2 groups: the preconditioning group (PCG), which contained 10 animals, and the hepatic pedicle was isolated and submitted to clamping with microvascular clamp (10 minutes of ischemia and 10 minutes of reperfusion, followed by 30 minutes of ischemia and 30 minutes of reperfusion); and the simulated operation group (SOG), which contained 10 animals submitted to manipulation of the hepatic pedicle and observation for the same length of time, with blood collected for transaminase dosage measurements, and liver biopsy for evaluation of mitochondrial respiration and histologic liver analysis and after sacrificed under anesthesia. The project was approved by the Ethics Committee on Animal Experimentation CEEA/UNICAMP under protocol number 3905-1. RESULT: The PCG mitochondria showed the same respiration level as the SOG, when stimulated with the addition of adenosine diphosphate or carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the respiratory control ratio and resting of velocity of respiration the groups behaved in a similar way. The PCG presented high aspartate and alanine transaminases (P < .03) and about 60% of sinusoidal congestion and venous congestion in the histologic analysis when compared with SOG. CONCLUSION: We found that ischemia with preconditioning in Wistar rats can lead to mild histologic and biochemical dysfunction without leading to impairment of mitochondrial respiration.

Immunoexpression of Heat Shock Protein 70, Glypican 3, Glutamine Synthetase, and Beta-Catenin in Hepatocellular Carcinoma After Liver Transplantation: Association Between Positive Glypican 3 and Beta-Catenin With the Presence of Larger Nodules.

BACKGROUND: Hepatocellular carcinoma (HCC) is the 6th leading cause of cancer worldwide. Its recurrence ranges from 6% to 26%. In the literature, many factors are associated with higher risk of recurrence, without a clear definition of the best method that could predict this highly lethal event. OBJECTIVE: The aim of this study was to evaluate the immunoexpression of immunohistochemical markers: HSP70, glypican 3, glutamine synthetase, and beta-catenin, as well as studying their association with tumor characteristics and prognosis of patients undergoing liver transplantation for HCC. METHODS: We studied 90 patients who underwent liver transplantation from 1998 to 2012. Afterwards we evaluated factors related to survival, tumor recurrence, and the correlation of expression of the immunohistochemical markers. RESULTS: Immunohistochemical marker glutamine synthetase showed a positive trend toward better survival. HSP70-positive patients had a higher prevalence of histologic grade III. Patients with positive glypican 3 showed larger lesions and a higher number with AFP >200 ng/mL. Patients with positive beta-catenin showed larger nodules and more with histologic grade III. The association between beta-catenin and glypican 3 showed positive association with larger nodules. CONCLUSIONS: Most of the markers studied had a correlation with at least one of the variables studied, confirming our hypothesis that these markers can indeed assist in assessing the prognosis of patients undergoing liver transplantation for HCC.

Survival benefits of interferon-based therapy in patients with recurrent hepatitis C after orthotopic liver transplantation.

Recurrent hepatitis C after orthotopic liver transplantation (OLT) is universal and can lead to graft failure and, consequently, reduced survival. Hepatitis C treatment can be used to prevent these detrimental outcomes. The aim of this study was to describe rates of hepatitis C recurrence and sustained virological response (SVR) to interferon-based treatment after OLT and its relationship to survival and progression of liver disease through retrospective analysis of medical records of 127 patients who underwent OLT due to cirrhosis or hepatocellular carcinoma secondary to chronic hepatitis C between January 2002 and December 2013. Fifty-six patients were diagnosed with recurrent disease, 42 started interferon-based therapy and 37 completed treatment. Demographic, treatment- and outcome-related variables were compared between SVR and non-responders (non-SVR). There was an overall 54.1% SVR rate with interferon-based therapies. SVR was associated with longer follow-up after treatment (median 66.5 vs 37 months for non-SVR, P=0.03) and after OLT (median 105 vs 72 months, P=0.074), and lower rates of disease progression (15 vs 64.7%, P=0.0028) and death (5 vs 35.3%, P=0.033). Regardless of the result of therapy (SVR or non-SVR), there was a significant difference between treated and untreated patients regarding the occurrence of death (P<0.001) and months of survival (P<0.001). Even with suboptimal interferon-based therapies (compared to the new direct-acting antivirals) there is a 54.1% SVR rate to treatment. SVR is associated with improved survival and reduced risks of clinical decompensation, loss of the liver graft and death.

Long-Time Choledochal Clamping in Wistar Rats Causes Biliary Obstruction Progressing to Hepatic Fibrosis.

BACKGROUND: Biliary complications are important causes of morbidity and mortality in patients undergoing hepatic surgery. The aim of the study was to evaluate late liver alterations after a long period of choledochal clamping in Wistar rats. METHODS: Ten male Wistar rats, weighing 304 grams, anesthetized with sodium thiopental (25 mg/kg) and xylazine (10 mg/kg) intravenously, were distributed into 2 groups: the choledochal clamping group (CCG) and the operation sham group (OSG), with 5 animals each submitted to an abdominal incision. In the CCG, the choledochal was isolated, dissected, and clamped with a microvascular clamp for 40 minutes. After this occlusion time, the clamp was removed and the incision was closed. In the OSG the animals, under normal conditions, were submitted only to anesthesia and laparotomy for choledochal manipulation. In all animals, after the 31st day, a hepatic biopsy was carried out for histology and blood biochemical tests: total bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transferase. The animals were euthanized under anesthesia. This research was approved by the Ethics Committee on Animal Use (CEUA, Unicamp, No. 2511-1). RESULTS: In the CCG, 100% of the animals showed bile duct dilatation, ductular proliferation, and portal inflammatory infiltrate; 60% showed regenerative nodule formation; and 80% had porta-porta septa and foci of necrosis, all of which were not found in the OSG. All CCG group biochemical tests had significant increases (P < .05) compared with OSG. CONCLUSIONS: Long-time choledochal clamping in Wistar rats caused hepatic dysfunction and biochemical and histological injuries with degrees of distortion to the hepatic architecture.

Clinical and pathological challenges in the diagnosis of late-onset biliary atresia: four case studies

Biliary atresia (BA) is classically described at the neonatal age. However, rare cases of BA in older infants have also been reported. We report four cases of late-onset BA in infants older than 4 weeks (3 males, 1 female), and describe the diagnostic and management difficulties. One of the cases had a late-onset (29 weeks) presentation with a successful surgical procedure. We highlight the importance of this unusual differential diagnosis in infants with cholestatic syndrome, who may benefit from Kasai surgery, regardless of age.

Clinical and pathological challenges in the diagnosis of late-onset biliary atresia: four case studies.

Biliary atresia (BA) is classically described at the neonatal age. However, rare cases of BA in older infants have also been reported. We report four cases of late-onset BA in infants older than 4 weeks (3 males, 1 female), and describe the diagnostic and management difficulties. One of the cases had a late-onset (29 weeks) presentation with a successful surgical procedure. We highlight the importance of this unusual differential diagnosis in infants with cholestatic syndrome, who may benefit from Kasai surgery, regardless of age.

No protective function found in Wistar rats submitted to long ischemia time and reperfusion after intermittent clamping of the total hepatic pedicle.

BACKGROUND: Although the intermittent Pringle maneuver is used for major transplant surgery, traumas, and hepatic protection, long ischemia time and reperfusion may limit some protection in Wistar rats. The aim of the study was to evaluate the protection effects of intermittent clamping in the total hepatic pedicle after a long period of ischemia and reperfusion in Wistar rats. METHODS: Forty-two male Wistar rats, weighing ± 327.7 g, were anesthetized intravenously with sodium thiopental and given a U-shaped incision in the abdomen. The total hepatic pedicle was isolated and subjected to clamping with a microvascular clamp. Groups included were the continuous group (CG, n = 14, 40 minutes of ischemia/40 minutes of reperfusion); the intermittent group (IG, n = 14, 4 cycles a 10 minute ischemia/reperfusion 10 minutes); and the sham group (SG, n = 14, 80 minutes of observation time). Blood collection for transaminase dosage was carried out, and hepatic biopsy specimens were taken for mitochondrial respiration and histological evaluation. RESULTS: In groups CG and IG, aspartate aminotransferase and alanine aminotransferase enzymes were elevated in comparison to group SG (P < .008); mitochondrias, when stimulated by use of adenosine diphosphate or carbonylcyanide p-trifluoromethoxyphenylhydrazone, had a significant decrease in mitochondrial respiration (P < .05), and the respiratory control ratio in the ischemic groups was lower (P < .03) when compared with the GS. On histological examination, 100% of the GC had lesions: 33% focal hemorrhagic necrosis, 17% sinuzoidal congestion and/or vacuolization, and 50% venous congestion; in the IG, 100% had lesions: 43% sinusoidal congestion and/or vacuolization and 57% venous congestion. CONCLUSIONS: The intermittent total hepatic pedicle clamping for a long period of time in the Wistar rats had no efficacy in protection of liver injury.

Late biliary obstruction in Wistar rats after intermittent hepatic pedicle clamping.

INTRODUCTION: The temporary vascular occlusion of hepatic flow is one of the essential procedures in hepatic surgery. AIM: Evaluate the late liver alterations after intermittent pedicle hepatic clamping (IHPC) in Wistar rats. METHODS: Male Wistar rats (n = 14) with average weight of 281.1 g, were anesthetized with intraperitoneal ketamine 5%. The IHPC group (n = 7) was submitted to U-shaped abdominal incision; the hepatic pedicle was isolated and submitted to IHPC ischemia 4 times, 5 minutes each, followed by reperfusion 4 times, 5 minutes each. The simulated operation group (n = 7) was subjected to anesthesia, laparotomy, and manipulation of the hepatic pedicle. On day 35, after fasting for 12 hours, liver biopsies were collected and blood was tested for liver aminotransferases (aspartate aminotransferase/alanine aminotransferase). RESULTS: All the IHPC group animals had a dilated common bile duct and increased liver enzymes (P < .05 by Mann-Whitney test). Ductular proliferation (100% of cases), porta-porta septa (42.8%), formation of lumps (42.8%), foci of necrosis (14.2%), and bile plugs (14, 2%) were observed only in the IHPC group. CONCLUSION: In rats, IHPC caused morphologic features leading to biliary obstruction.

Liver transplantation in a patient with clinical manifestations of cryptogenic cirrhosis: a case report of hepar lobatum as a primary liver condition.

BACKGROUND: This article reports a case of hepar lobatum, a peculiar and rare type of liver deformity, originally described in association with infectious or parasitic diseases and with malignancies. CASE REPORT: We have described a 42-year-old woman with this disorder, which was unrelated to the known conditions and referred for liver transplantation for having clinical manifestations of cirrhosis, portal hypertension, and impaired hepatic function. CONCLUSIONS: The observed histologic pattern suggests that hepar lobatum could be, in some patients, the effect of a primary process of hamartomatous origin involving the organ vascular supply.

Liver failure and the need for transplantation in 6 patients with hepatoportal sclerosis.

Hepatoportal sclerosis (HPS), first reported by Mikkelsen et al in 1965, is a pathologic condition that does not cause cirrhotic portal hypertension. The primary hepatic lesion in HPS is found in portal vein branches with preserved synthetic function. Rarely do patients with HPS need liver transplantation. The aim of this study was to describe the clinical and pathologic features of 6 HPS cases who underwent liver transplantation (OLT). From 2000 to 2008, 6 OLT candidates were diagnosed with HPS: 3 displayed bleeding varices and 4 ascites. Child-Pugh evaluation was class B (n = 4) or C (n = 2). The Model for End-stage Liver Disease scores were 18 (n = 2), 20 (n = 3), and 22 (n = 1). Cirrhosis resulted from presumed diagnoses of alcohol n = (1), autoimmune n = (2) or cryptogenic cirrhosis n = (3). On histologic examination, there was marked phlebosclerosis in all cases, including nonocclusive portal vein thrombosis (n = 3), intense portal fibrosis (n = 1), moderate portal fibrosis (n = 5), and uniform moderate sinusoidal dilatation without megasinusoid formation, but with ductal biliary proliferation and ductal biliary fibrosis in all cases. Cholestasis was observed in 1 and incomplete septal cirrhosis in 4 cases. None of the subjects showed histological features of the presumed underlying liver disease. The overall survival of this group was no different from that of other OLT patients. HPS causing hepatic failure may require liver transplantation. Fhlebosclerosis andportal fibrosis may contribute to the loss of hepatic synthesis leading to the need for hepatic transplant. Significant portal fibrosis and phlebosclerosis can contribute to hepatic parenchymal and posterior synthetic loss.

Biliary obstruction after short-time choledochal clamping in rats.

INTRODUCTION: Biliary complications after liver procedures can lead to morbidity and poor survival. AIM: The aim of this study was to evaluate the hepatic function after quick clamping of the common bile duct (BD) in Wistar rats. METHODS: Twelve male Wistar rats with a mean weight of 323.14 g were anesthetized with sodium thiopental intravenous (IV). The common BD Clamping Group (BDCG; n = 6) was submitted to an abdominal incision (2 cm); the BD was isolated, dissected, and underwent clamping for 10 minutes with a microvascular clamp. After this time, the clamp was removed and the incision closed. The Sham Operation Group (SOG; n = 6 rats), under normal conditions, were subjected only to anesthesia and laparotomy and later control tests. On the 28th day liver and choledoch biopsy and biochemical tests were performed on all animals: total bilirubin (TB), alkaline phosphatase (ALK-P), aminotransferases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]), and gamma-glutamyl transferase (GGT). After the tests all the rats were humanely killed while still under anesthesia. RESULTS: In this study 83% of the animals in the BDCG had large dilatation of the common BD with ductular proliferation, formation of septae, as well as multiple foci of parenchymal necrosis including micro-abscess formation. We also observed alterations in biochemical tests (P < .05). CONCLUSION: Our study demonstrated that BD clamping even for a short time was sufficient to generate important morphological alterations in the liver and BD, as confirmed by enzymatic and histological analysis. Therefore, this technique can be used as a model of biliary obstruction for future studies.

Predicting survival after liver transplantation using up-to-seven criteria in patients with hepatocellular carcinoma.

BACKGROUND: Liver transplantation is a controversial issue in the initial management of hepatocellular carcinoma (HCC). The aim of this study was to demonstrate that up-to-seven could be useful as a model in evaluating potential candidates for liver transplantation for HCC. METHODS: Between January 1997 and December 2010, 84 patients undergoing liver transplantation were analyzed for HCC. Pathologic variables included the number of lesions, maximal tumor diameter, vascular invasion, and tumor grade. The following variables were recorded: Age, gender, recurrence of HCC, survival, donor age, incidental, Milan criteria, Model for End-Stage Liver Disease score, and alpha-fetoprotein (AFP) levels. To evaluate variables we used the Kaplan-Meier method, Cox regression and Kolmogorov-Smirnov test. RESULTS: There were 68 (80.9%) patients within up-to-seven criteria and 16 (19.1%) beyond this criteria. AFP levels were 96.4 versus 464.3 ng/dL in patients within versus beyond up-to-seven criteria. The median diameter of the largest nodule was 22.2 versus 32.0 mm in patients within versus beyond up-to-seven criteria, respectively. The median sum of nodules was 1.8 and 5.6 nodules in patients within versus beyond up-to-seven criteria. Vascular invasion was present in 11 (13.0%) patients within versus 7 (8.3%) beyond. Recurrence was observed in 9 (10.7%) patients and only 3 were beyond up-to-seven criteria, 2 had vascular invasion, and another showed poor differentiation. Post liver transplantation survival rates were 87.7%, 74.5%, and 65.3% at 1, 3, and 5 years in patients within up-to-seven versus 80.7%, 51.7%, and 32.1% beyond (P = .03). Similar results were observed when we applied Milan criteria or expanded Milan criteria. The largest nodule was the only predictive factor after proportional hazard Cox regression application (Beta = 0.037; exponent = 1.0377; Wald = 4.542; P = .03). In the present study, up-to-seven criteria could be useful to evaluate potential liver transplant candidates due to HCC.

Prognostic factors for hepatocellular carcinoma recurrence: experience with 83 liver transplantation patients.

INTRODUCTION: Orthotopic liver transplantation (OLT) is a rational therapeutic option for early-stage hepatocellular carcinoma (HCC) providing a potential cure and improving survival. METHODS: This retrospective study of a longitudinal cohort used an electronic database collected prospectively from September 1997 to May 2010. The variables were gender, age (years), and alpha-fetoprotein (AFP) level (ng/mL). In explanted livers we observed: microvascular or macrovascular invasion, number of nodules and their largest size, Edmondson-Steiner histological differentiation, incidental tumor transarterial chemoembolization (TACE), Milan criteria, and previous down-staging. RESULTS: Five of 83 (6.0%) subjects including 68 (82%) males with a mean time to diagnosis of 9 months experienced tumor relapses. Mean patient age at HCC recurrence was 55.3 years for male and 44.6 years for female subjects. Vascular invasion was detected in 17/83 (20.5%) subjects, namely 2% of macrovascular invasion, and 52.5% with expanded Milan criteria due to an increased number and size of nodules in the explanted livers. An incidental tumor was observed in 29.5% of cases. Preoperative TACE treatment was performed in 13 (15.6%) patients. None of the patients who had a HCC recurrence had undergone TACE. AFP level at the time of recurrence was around 1,900 ng/mL. The predictive factor for mortality was nodule size (P=.04; hazard ratio=0.0269; confidence interval [CI], 95% 0.0094-0.299). CONCLUSION: Patients with relapses showed the worst survival and tumor size was a predictive factor for recurrence.

Large benign hepatocellular nodules in cirrhosis due to chronic venous outflow obstruction: diagnostic confusion with hepatocellular carcinoma.

Budd-Chiari syndrome (BCS) in patients progressing to cirrhosis is an indication for liver transplantation. At this stage of disease, it is common to find large benign hepatocellular nodules (LBHNs) of undetermined cause that may be confused with hepatocellular carcinoma (HCC). Patients with indications for liver transplantation are currently classified according to the MELD (Model for End-Stage Liver Disease) severity score. When they fit Barcelona and Milan eligibility criteria for HCC, they receive 20 points. Thus, misdiagnosis of HCC leads to a privileged position on the waiting list. Herein, we have reported three BCS cases of cirrhotic patients who underwent liver transplantation; the pathologic results of their explanted livers showed LBHN. We analyzed three of 489 OLT who had chronic venous outflow obstruction (CVOO) the first case: was a 19-year-old man, with BCS of undetermined cause. The second 20-year-old female patients displayed BCS due to antiphospholipid syndrome the third, 45-year-old man had CVOO diagnosed preliminarily due to cryptogenic cirrhosis in the explanted liver. In the three cases, the nodules in the explant measured 0.5 to 2.4 cm. In the first case, the diagnosis was not in doubt; in the second case, 23 nodules were confused with HCC histologic evaluation, and in the third case three larger hypervascular nodules were misdiagnosed as HCC in the preoperative period despite low alpha-fetoprotein levels. In conclusion it is fundamental to recognize these benign lesions so as to avoid misdiagnosis, thereby allowing the proper selection of candidates for liver transplantation.

Expanded Milan criteria on pathological examination after liver transplantation: analysis of preoperative data.

BACKGROUND: We sought to evaluate the accuracy of imaging techniques related to the Milan criteria (MC) compared with the explant histology and the survival of these patients. METHODS: Between 1997 and 2006, we selected 45 cirrhotic patients with hepatocellular carcinoma distributed into two groups according to explant histology: MC and Expanded Milan Criteria (EMC). Age, gender, preoperative imaging (ultrasound [US] and/or computed tomography [CT]), maximal tumor dimension, number of tumors, explanted histology, histology degree, alpha-fetoprotein (AFP) level and vascular invasion were compared among the patients to evaluate the value of these prognostic factors for survival after liver transplantation. RESULTS: By histology 42.2% explants were identified as EMC. The mean AFP level was 204.5 ng/mL. Vascular invasion was detected in 31.5% of explants and 68.4% showed incidental tumors. The survival rates after 10 years were 47.4% whereas MC patients showed 57.77%. The mean AFP level among MC patients was 150.2 ng/mL with vascular invasion detected in 7.7% of explants, and 47.4% with incidental tumors. The overall sensitivity of the imaging techniques was 83.3% for CT and 75% for US. The specificity was 96% for CT and 80.1% for US. CONCLUSION: Scan examinations in the preoperative evaluation underestimated about 42.2% of tumors. Those patients had vascular invasion but the survival after 10 years was similar between the ECM and MC groups.

Can the use of marginal liver donors change recipient survival rate?

Liver transplantation as a therapeutic method for the treatment of end-stage liver disease is beclouded by a scarcity of organs. The aim of this study was to retrospectively analyze the relation between the classification of donors as marginal versus ideal and recipients survival after 148 of 197 orthotopic liver transplantations (OLT) performed from 1991 to 2001. Donors were classified as marginal if they showed the major criteria of: age over 55 years, aspartate aminotransferase greater than 150 UI/L; serum bilirubin greater than 2 mg/dL, serum sodium greater 150 mEq/L, high-dose dopamine or any other vasoactive amine, cardiac arrest, intensive care unit (ICU) stay over 5 days, and moderate severe macrosteatosis. The minor criteria for a marginal donor were: use of dopamine below 10 microg/kg/min, history of alcoholism, drug abuse, ICU stays less than 4 days, microsteatosis of any degree, and mild macrosteatosis. Statistical analysis was performed using Cox regression analyzing and the Kaplan-Meier survival method. The rate of marginal donors was 61.5%. The 180 postoperative day survival was 77.0%. Survival rates were 81.1% for recipients of marginal donor organs, and 70.7% for ideal donor recipients (P >.05). In conclusion, the use of marginal liver donors is viable and safely expands the numbers of liver transplants, thereby diminishing the number of waiting list deaths.